1. Field of the Invention
The present invention relates to biofilm inhibitors and, more particularly, squarylated lactones for inhibiting quorum sensing.
2. Description of the Related Art
The term quorum sensing (QS) refers to the ability of a microorganism to sense and monitor the presence of other microbes by producing and responding to signal molecules called autoinducers (also AI). QS regulates microbial population levels and is a critical determinant of biological processes like biofilm formation, luminescence or pathogenesis that is crucial for the microbial growth and survival. This cell-to-cell communication is carried out by signals such as N-acyl-L-homoserine lactones (AHLs) that bind to their cognate receptors when critical cell density is reached leading to optimal physiological and communal response.
QS generally govern multi-cellular behavior like bioluminescence, secretion of virulence factors, biofilm formation, sporulation, conjugation and pigment production. Initially, QS was considered to be a mechanism by which microbial cells count each other. But, recent evidence suggests that QS process can be induced by even a few cells confined to a very small volume. Generally, each species of bacteria tend to produce and detect signaling molecules that are responsible for their multi-cellular behavior. Among prokaryotes, the Gram negative bacteria use, extensively, signal molecules that have acyl homoserine lactones (also called autoinducer 1; AHLs) moieties in their structure. These signals effectively enter the cells, upon quorum, via diffusion and interact with their corresponding intracellular effectors. Upon reaching threshold concentrations these small molecules are detected by the cognate cytoplasmic LuxR-type proteins, which bind their corresponding autoinducers, and further bind to DNA to activate transcription of preferred target genes that may produce biofilm or virulence. The specificity of LuxR-AHL interactions is determined by the ability of the AHL signal's acyl chain to fit the hydrophobic binding pocket of the LuxR protein. Apart from the AHLs and the peptide-based signaling molecules, interspecies communication is facilitated by molecules called AI-2 (autoinducer 2).
The AI-2 is a generic name for a family of signal molecules having a basic furanosyl borate diester structure. All these molecules have a common precursor, 4,5-dihydroxy-2,3-pentanedione (DPD) that is generated by the LuxS enzyme. QS in E. coli has evoked significant interest leading to the discovery of different intercellular signaling pathways including those mediated by LuxR homolog SdiA, a LuxS/AI-2 system consisting of proteins LsrR and LsrK, an unknown AI-3 system and an indole mediated signaling system. There is lack of clarity regarding the QS apparatus in E. coli due to the incomplete availability of QS components. Moreover, some of the putative QS signals are intertwined with metabolism and it is not entirely possible to dissect the role of QS signals in both these processes. The AI-2 signal is considered to be responsible for interspecies communication in both Gram positive and Gram negative bacteria. For instance, E. coli detects and responds to AI-2 secreted by V. harveyi to assess changes in its cell population. DPD rearranges spontaneously into a family of AI-2 signals that have been attributed to cause formations of biofilms in E. coli. 
Biofilms constitute a community of microorganisms in which the cells are attached to the surface by means of a self-produced matrix called extracellular polymeric substance (EPS) or “slime”. The polymeric matrix is derived of proteins, nucleic acids and polysaccharides. Biofilms can form on biotic or abiotic surfaces and are prevalent in nature or in an industrial and hospital setting. The microbial cells existing in a planktonic form or in biofilms are found to vary physiologically and display differential genetic expressions.
Managing biofilms is currently a major area of interest as biofilms have huge implications in the health care industry. According to the United States National Institutes of Health, about 80% of chronic infections are biofilm related. The biofilm generally includes several bacterial species and as it gets thicker, the matrix assumes a complex structure and becomes impenetrable to the antibiotics. Biofilms routinely foul up medical instruments like catheters and implants, occur as dental plaques, lead to chronic ailments and persistent lung infections in cystic fibrosis patients that are mediated by Pseudomonas aeruginosa and are suspects in variety of diseases like prostatitis, endocarditis, and conjunctivitis. Researchers are just beginning to understand that bacterial cells in biofilms are 10-1000 times more resistant to antibiotics than in their planktonic counterparts. Destroying the bacterial cells in biofilms in exceedingly difficult due to the nature of the matrix and also because the bacterial cell behavior is changed. Apparently, bacterial cells in a biofilm have lower metabolism, are relatively quiescent and are rarely dividing. Most anti-bacterials unfortunately target dividing cells and are aimed at metabolism, DNA or protein synthesis.
Due to the considerable medical problems posed by biofilms there is a continued need and desire to develop newer antimicrobial tools that target not merely biofilms but the processes that lead to the formation of biofilms. Emergence of newer multi-drug resistant strains of microbes has made the development of novel approaches and tools to treat microbial diseases a top priority. Using chemical methods, the first approach could target the QS process itself causing quenching of cell-to-cell communication leading to prevention of multicellular behavior like biofilm formation and virulence. For instance, developing autoinducer analogs that block the LuxR, LuxS or LuxI-type proteins and inhibit their activities would be of great interest. The second approach could entail the use of enzymes to enable degradation of AHLs. For instance, lactonase AiiA from A. tumefaciens degrades AHLs and the lactonase AiiA from Bacillus species can hydrolyze the AHLs lactone ring to acyl homoserine, an ineffective QS signal, and prevent E. caratovora to cause soft rot disease in plants. The third approach could potentially target the AHL biosynthetic pathway, encouraging interruption in AHL production by use of synthetic analogs of AHL precursors. Recently antibodies and natural products have been used to break down QS and biofilm development in Gram-negative bacteria. On the other hand, with surfaces the incentive is to develop novel bio-inert surface chemistry tools that would inhibit the cell adhesion process itself thereby eliminating any possibility of biofilm formation.
Over the past 20 years or so, a significant amount of research has been directed towards the design and synthesis of ligands that disrupt AHL binding to its cognate receptors and inhibit QS. A variety of non-natural systems including synthetic mimics of marine natural products and modified AHLs have been developed primarily to modulate QS in microbial populations, but there still exists a scarcity of non-toxic inhibitors of QS and biofilm formation. New design and synthetic strategies are clearly needed to expand the current set of quorum-sensing modulators in Gram negative bacteria. Designing new quorum sensing modulators has been an increasing challenge because the few synthetically developed antagonists and agonists of quorum sensing have diverse structures and their mechanism of action is unclear. Moreover, as multiple microbial species generally co-exist together there is a significant motivation to develop chemical signal entities that can either selectively target or display broad-range activity against multiple AHL-receptor proteins that are available. At this juncture, it is interesting to note that the homology of the putative binding sites of the ˜50 well-known AHL-receptor proteins (70-80%) suggests that if non-natural mimics of AHL target these sites then both selective and broad-spectrum molecules could be developed.